Periodontal diseases are prevalent destructive oral polymicrobial infections that remain a significant public health burden. Porphyromonas gingivalis is a gram (-) anaerobe and a major etiological agent in severe forms of the disease. The organism is a successful colonizer of gingival epithelial cells (GECs), which form an initial defense to invading pathogens while serving as primary target cells for opportunistic bacteria. P. gingivalis is a host-adapted pathogen that can survive and replicate in primary GECs, and later spread intercellularly. The infection inhibits GEC death by impacting mitochondrial-apoptotic pathways and suppressing ATP-P2X7 receptor signaling. Ligation of P2X7 receptors with the danger signal, extracellular ATP (eATP), has recently been shown to result in production of intracellular reactive-oxygen-species (ROS) in macrophages. Although previously known mainly as a toxic agent, ROS are increasingly recognized to play an important physiological role by modulating a variety of key cellular functions including apoptosis, immune responses, and intracellular infections. P. gingivalis secretes an effector, nucleoside diphosphate kinase (Ndk), which scavenges eATP and diminishes P2X7 activity. We have recently found that P. gingivalis modulates cytosolic ROS production and subsequently blocks eATP-induced oxidative stress in primary GECs via secretion of Ndk during infection. However, the Ndk-deficient mutant of P. gingivalis lacks the ability to inactivate eATP-induced oxidative stress and persist intracellularly. The eATP-induced ROS generation appears to be mediated by P2X7 receptor signaling coupled with NADPH oxidase and mitochondrial oxidative stress pathways. The goal of this study is: to define the basic host mechanisms which P. gingivalis utilizes to inhibit eATP-induced cellular oxidative stress and to characterize the role of Ndk in targeting specific host metabolic and regulatory pathways potentially critical for the organism's survival and evasion of eATP- mediated intracellular killing. The kinetics and source of the ROS in primary GECs will be determined by flow-cytofluorimetry, fluorescence cell imaging, and high-resolution Oxygraph. We will identify infection- targeted host molecular circuitries and characterize the spatio-temporal secretion and function of Ndk employing a combination of selective inhibitors, agonists, gene depletion by siRNA, q-PCR, immuno- biochemical and fluorescent protein reporter systems in conjunction with confocal quantitative-image analyses. Finally, we will elucidate manipulation of eATP signaling by P. gingivalis for intracellular growth and survival. These studies will provide a detailed characterization of previously unexplored host molecular networks targeted by Ndk, an effector of P. gingivalis, for persistence in gingival epithelium. The knowledge gained may translate into the development of specific physiological inhibitors that may control or reduce the severity of chronic infections caused by this opportunistic pathogen. PUBLIC HEALTH RELEVANCE: P. gingivalis is a major contributor to severe forms of periodontal diseases, which are among the most common chronic infections affecting the U.S. population. The proposed research aims to define the basic complex molecular events between P. gingivalis and human gingival cells that are infected by this pathogen. These studies will provide valuable data needed to develop specific therapeutic agents to subvert unfavorable interactions with host cells and control the growth and colonization of this microorganism in oral tissues.